SummaryThe impact of neonicotinoid insecticides on the health of bee pollinators is a topic of intensive research and considerable current debate [1]. As insecticides, certain neonicotinoids, i.e., N-nitroguanidine compounds such as imidacloprid and thiamethoxam, are as intrinsically toxic to bees as to the insect pests they target. However, this is not the case for all neonicotinoids, with honeybees orders of magnitude less sensitive to N-cyanoamidine compounds such as thiacloprid [2]. Although previous work has suggested that this is due to rapid metabolism of these compounds [2, 3, 4, 5], the specific gene(s) or enzyme(s) involved remain unknown. Here, we show that the sensitivity of the two most economically important bee species to neonicotinoids is determined by cytochrome P450s of the CYP9Q subfamily. Radioligand binding and inhibitor assays showed that variation in honeybee sensitivity to N-nitroguanidine and N-cyanoamidine neonicotinoids does not reside in differences in their affinity for the receptor but rather in divergent metabolism by P450s. Functional expression of the entire CYP3 clade of P450s from honeybees identified a single P450, CYP9Q3, that metabolizes thiacloprid with high efficiency but has little activity against imidacloprid. We demonstrate that bumble bees also exhibit profound differences in their sensitivity to different neonicotinoids, and we identify CYP9Q4 as a functional ortholog of honeybee CYP9Q3 and a key metabolic determinant of neonicotinoid sensitivity in this species. Our results demonstrate that bee pollinators are equipped with biochemical defense systems that define their sensitivity to insecticides and this knowledge can be leveraged to safeguard bee health.

SummaryGene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3, 4, 5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6, 7, 8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait.

The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis , the most abundant and economically important solitary bee species in Central Europe. We show that O . bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N -cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O . bicornis to N -cyanoamidine and N -nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo . Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators.

Host shifts can lead to ecological speciation and the emergence of new pests and pathogens. However, the mutational events that facilitate the exploitation of novel hosts are poorly understood. Here, we characterize an adaptive walk underpinning the host shift of the aphid Myzus persicae to tobacco, including evolution of mechanisms that overcame tobacco chemical defenses. A series of mutational events added as many as 1.5 million nucleotides to the genome of the tobacco-adapted subspecies, M. p. nicotianae, and yielded profound increases in expression of an enzyme that efficiently detoxifies nicotine, both in aphid gut tissue and in the bacteriocytes housing the obligate aphid symbiont Buchnera aphidicola. This dual evolutionary solution overcame the challenge of preserving fitness of a mutualistic symbiosis during adaptation to a toxic novel host. Our results reveal the intricate processes by which genetic novelty can arise and drive the evolution of key innovations required for ecological adaptation.

1. Pesticide exposure has been implicated as a contributor to insect pollinator declines. In social bees, which are crucial pollination service providers, the effect of low-level chronic exposure is typically non-lethal leading researchers to consider whether exposure induces sub-lethal effects on behaviour and whether such impairment can affect colony development. 2. Studies under laboratory conditions can control levels of pesticide exposure and elucidate causative effects, but are often criticised for being unrealistic. In contrast, field studies can monitor bee responses under a more realistic pesticide exposure landscape; yet typically such findings are limited to correlative results, and can lack true controls or sufficient replication. We attempt to bridge this gap by exposing bumblebees to known amounts of pesticides when colonies are placed in the field. 3. Using 20 bumblebee colonies, we assess the consequences of exposure to the neonicotinoid clothianidin, provided in sucrose at a concentration of five parts per billion, over five weeks. We monitored foraging patterns and pollen collecting performance from 3282 bouts using either a non-invasive photographic assessment, or by extracting the pollen from returning foragers. We also conducted a full colony census at the beginning and end of the experiment. 4. In contrast to studies on other neonicotinoids, showing clear impairment to foraging behaviours, we detected only subtle changes to patterns of foraging activity and pollen foraging during the course of the experiment. However, our colony census measures showed a more pronounced effect of exposure, with fewer adult workers and sexuals in treated colonies after five weeks. 5. Synthesis and applications. Pesticide induced impairments on colony development and foraging could impact on the pollination service that bees provide. Therefore our findings, that bees show subtle changes in foraging behaviour and reductions in colony size after exposure to a common pesticide, has important implications and helps to inform the debate over whether the benefits of systemic pesticide application to flowering crops outweigh the costs. We propose that our methodology is an important advance to previous semi-field methods and should be considered when considering improvements to current ecotoxicological guidelines for pesticide risk assessment